DE69316903T2 - SAINTOPINE DERIVATIVES - Google Patents

Abstract

A saintopin E derivative represented by general formula (I) and useful as an antibacterial and antitumor agent, wherein when R<1> represents hydrogen, then R<2> represents SO2OH (in the case of UCE1022), while when R<1> represents acetyl, then R<2> represents hydrogen (in the case of saintopin E). <CHEM>

Description

The present invention relates to Saintopin derivatives which possess antibacterial and antitumor activity and are therefore useful as antibacterial and antitumor agents.

It is already known that compounds with an anthraquinone backbone, such as anthracycline, have antibiotic activity [CRC Handbook of Antibiotic Compounds, 3 (1981) 61].

From the compound represented by formula A UCT 1003 (Saintopin)

is also known to have antitumor activity [Biochemistry, 30, (1991) 5838 -5845 and unexamined published Japanese Patent Application No.200655/90].

In accordance with the present invention, further Saintopin derivatives were found to have antibacterial and antitumor activity.

These are compounds of the formula:

in which R¹ is a hydrogen atom and R² is SO₂OH, or R¹ is acetyl and R² is a hydrogen atom.

Hereinafter, the compound in which R¹ is hydrogen and R² is SO₂OH is referred to as UCE 1022 and the compound in which R¹ is acetyl and R² is hydrogen is referred to as Saintopin E.

The compounds UCE 1022 and Saintopin E are produced by microorganisms of the genus Paecilomyces and can be obtained from their cultures.

The compound UCE 1022 has the following physico-chemical properties.

(1) Molecular weight: 418

(2) Molecular formula: C18 H10 O10 S

(3) Mass spectrum: secondary ion mass spectrum, negative mode

(Matrix: m-nitrobenzyl alcohol): m/z: 417 (M-H)&supmin;

High-resolution FAB mass spectrum, negative mode

(Matrix: m-nitrobenzyl alcohol): m/z

Found: 416,9919

Calculated for C₁₈H₁₀O₁₀S: 416.9917

(4) UV absorption spectrum: (measured in methanol) λmax nm, (E): 241(22,000), 274 (27,500), 306 (16,100), 339 (9,800), 483 (12,300)

(5) IR absorption spectrum (measured by the KBr method):

νmax cm4: 3360, 3210, 1625, 1600, 1400, 1330, 1265, 1235, 1045

(6) 13C NMR spectrum (125 MHz, methanol-d4 solution): δ ppm: 108.3; 109.2; 109.3; 111.4; 111.6; 114.8; 116.3; 121.9; 130.0; 137.5; 141.2; 154.3; 161.9; 166.4; 166.5; 166.6; 183.4; 190.8

(7) 1H NMR spectrum (500 MHz, methanol-d4 solution): δ ppm: 6.57 (1H, d); 7.09 (1H, d); 7.21 (1H, d); 7.41 (1H, d); 7.96 (1H, s)

(8) Solubility: soluble in water, methanol and ethanol, and moderately soluble in n-hexane (UCT 1003 is moderately soluble in water)

(9) Colour reaction: Positive in iodine and cerium sulphate sulphuric acid tests

(10) Colour and appearance of the substance: reddish-purple powder

(11) Thin layer chromatography: In thin layer chromatography on silica gel (HPTLC plate Art. 5715, manufactured by Merck & Co., Inc.) using a solvent composed of n-hexane: ethyl acetate: methanol: acetic acid (6:4:1:1, v/v), The Rf value of the mobile phase is 0.25 (the point of UCE 1022 is detectable by absorption in the visible and UV range).

Saintopin E has the following physicochemical properties.

(1) Molecular weight: 380

(2) Molecular formula: C&sub2;&sub0;H&sub1;&sub2;O&sub8;

(3) Mass spectrum: High-resolution FAB mass spectrum

(Matrix: m-nitrobenzyl alcohol): m/z:

Found: 381.3223 (M+H)&spplus;

Calculated for C₂₀H₁₂O₈:381.3214

(4) UV absorption spectrum (measured in methanol) λmax nm (ε); 290 (17,100), 338 (8,100), 566 (7,300)

(5) IR absorption spectrum (measured by the KBr method):

νmaxcm⁻¹: 3431,3230,1622,1564, 1437,1387,1269,1163

(6) 1H NMR spectrum (500 MHz, DMSO-d6 solution): δ ppm: 14.10 (1H, br,s), 13.43 (1H, br,s), 10.76 (1H, br,s), 7.46 (1H, s), 7.04 (1H, d, 2.1 Hz), 6.53 (1H, d, 2.1 Hz), 6.34 (1H, s), 2.66 (3H, s)

(7) Solubility: slightly soluble in dimethyl sulfoxide (DMSO), methanol and acetone, soluble in chloroform and ethyl acetate, and moderately soluble in water and n-hexane

(8) Colour and appearance of the substance: bluish-purple powder

(9) Thin layer chromatography: In thin layer chromatography on silica gel (HPTLC plate Art. 5715, manufactured by Merck & Co., Inc.) using a mobile phase composed of n-hexane: ethyl acetate: methanol: acetic acid (6:4:1:1, v/v), the Rf value is 0.50.

(10) High performance liquid chromatography: retention time: 8.56 min. (column: YMC AM312 ODC, manufactured by YMC Corporation; flow rate: 1 mL/min.; mobile phase: a 70% methanol solution containing 5 mM ammonium acetate)

The biological activities of the compounds (I) are described below.

Test example 1: Antibacterial effectiveness

The minimum inhibitory concentration (MIC) against the growth of various bacteria is shown in Table 1. The antibacterial activity was determined by the agar dilution method using a medium (13H value 7) comprising 3 g/L Bacto-Tryptone (manufactured by Difco Laboratories), 3 g/L meat extract, 1 g/L yeast extract, 1 g/L glucose and 16 g/L agar. Table 1

Test example 2: Antitumor activity against HeLaS₃ cells

HeLaS3 cells (ATCC HTB22) were suspended in a medium containing 10% fetal calf serum, 2 mM glutamine and MEM medium (manufactured by Nippon Pharmaceutical Co., Ltd.) (hereinafter referred to as Medium A) at a concentration of 3 x 104 cells/ml. The cell suspension was added to the wells of a 96-well microtiter plate at a rate of 0.1 ml per well. The cells in the plate were cultured in a CO2 incubator at 37 ºC for 20 hours. Subsequently, the test compound appropriately diluted with Medium A was added to the wells at a rate of 0.1 ml/well. The cells were further cultured in the CO2 incubator at 37 ºC for 72 hours, and then the supernatant solution was removed. To the residue was added a medium comprising Medium A and 0.02% Neutral Red in an amount of 0.1 ml per well, followed by culturing at 37 ºC in a CO₂ incubator for 1 hour, whereby the cells were stained. After removing the supernatant solution, the residue was washed once with physiological saline. The pigment was extracted with 0.001 N hydrochloric acid/30% ethanol, and the absorbance at 550 nm was measured using a microplate reader. The concentration of the test compound at which the cell growth was inhibited by 50% (1C50) was determined by comparing the Absorbance of untreated cells was calculated with that of cells treated with the test compound at known concentrations. The result is shown in Table 2. Table 2

Test example 3: Inhibition of growth against BALB3T3/H-ras cells

BALB 3T3/H-ras cells were suspended in F10 medium containing 10% fetal calf serum, 100 units/ml penicillin and 100 mg/ml streptomycin (a product of GIBCO Company; hereinafter referred to as Medium B) at a concentration of 2 x 10⁴ cells/ml. The cell suspension was added to wells of a 96-well microtiter plate at a rate of 0.1 ml per well. The cells in the plate were cultured in a CO₂ incubator at 37 ºC for 20 hours. Subsequently, the test compound appropriately diluted with Medium B was added to the wells at a rate of 0.05 ml per well. The cells were further cultured in the CO₂ incubator at 37 ºC for 72 hours. The system was treated in the same manner as in Test Example 2 to calculate the concentration of the test compound at which the growth of cells was inhibited by 50% (IC₅₀). The result is shown in Table 3. Table 3

The process for preparing the compounds (I) is described below.

UCE 1022 contained in the compounds (I) can be obtained by culturing a microorganism belonging to the genus Paecilomyces and having the ability to produce UCE 1022 in a medium, allowing UCE 1022 to accumulate in the culture, and recovering UCE 1022 from the culture.

As the UCE 1022-producing strains of the present invention, any strains belonging to the genus Paecilomyces and having the ability to produce UCE 1022 can be used. Furthermore, any mutations of such strains obtained by various artificial mutation methods such as UV irradiation, X-rays and treatment with mutagenic agents or by spontaneous mutation can also be used in the present invention, as long as they have the ability to produce UCE 1022. A typical example of a suitable strain is the UOE 1022 strain isolated from soil by the present inventors.

The mycological properties of strain UOE 1022 are as follows.

1. Macroscopic view

When strain UOE 1022 is cultured at 25 ºC on an agaric medium made of malt extract, the diameter of a colony reaches about 28 mm on the seventh day after the start of culture. The surface of the colony is flaky and has a white color. The center of the back of the colony has an orange color, and its edge has a pale yellow color. Dissolution of a soluble pale yellow pigment is observed in the culture medium.

When this strain is cultured at 25 ºC on a potato glucose agar medium, the diameter of a colony reaches 26 to 27 mm on the seventh day after the start of culture. The surface of the colony is flaky and its center has a purple or purple color and its edge has a white to gray color. The back of the colony has a lemon color. Dissolution of a soluble pale yellow pigment is observed in the culture medium.

The optimum growth temperature for this strain is in the range of 11 to 34ºC, and most preferably around 23ºC. The range of pH at which the strain can grow is 3 to 10.5, and the pH for optimum growth is around 6.

2. Viewing under an optical microscope

The strain UOE 1022 is cultivated at 25 ºC on agar medium of malt extract and is observed under the optical microscope. The results are as follows.

The hyphae are septate and smooth and branch well. The conidiophores arise from the hyphae, are colorless and smooth, and branch in the upper part in a whorled form or irregularly. At the tip of each branch of the Two or four phialides form at the conidiophores. In some cases one to four phialides form directly at the tip of each conidiophore. The phialides are colorless, smooth, lageniform, and taper to a point. They are 7 to 12 µm long and 2 to 3 µm wide near their junction, and taper to about 0.5 µm wide. Conidial ontogeny is enteroblastic. The phialoconidia are unicellular and fusiform or limoniform in shape. They are colorless and smooth and are 3 to 4.5 µm long and 1.5 to 2.5 µm wide. The conidia develop in the form of a long chain from the tip of the phialide. In this strain only the anamorph as described above is observed, with no teleomorph being observed at all.

A taxonomic study of this strain based on the above mycological properties according to "The Genera of Fungi Sporulating in Pure Culture, 2nd ed., Cramer, Vaduz; J. A. van Arx, 1974" revealed that it belongs to the genus Paecilomyces, which falls under the Hyphomycetes.

The strain was named Paecilomyces sp. JOE 1022 by the present inventors and deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology under accession number FERM BP-4066 on November 5, 1992.

The manufacturing process for Saintopin E is described below.

Saintopin E can be obtained by culturing a microorganism belonging to the genus Paecilomyces and having the ability to produce Saintopin E in a medium, allowing Saintopin E to accumulate in the culture, and recovering Saintopin E from the culture.

As the Saintopin E-producing strains of the present invention, any strains belonging to the genus Paecilomyces and having the ability to produce Saintopin E can be used. Furthermore, any mutations of such strains obtained by various artificial mutation methods such as UV irradiation, X-rays and treatment with mutagenic agents or by spontaneous mutation can also be used in the present invention, as long as they have the ability to produce Saintopin E. A typical example of a suitable strain is the strain SPC-13780.

The strain was obtained from the Fermentation Research Institute, Agency of Industrial Science and Technology under the Budapest Treaty with the accession number FERM BP-2256 (Date of original filing: January 24, 1989) (unexamined published Japanese Patent Application No.200655/90).

The culture procedures for UCE 1022-producing and Saintopin E-producing strains are as follows.

For culturing the strains used in the present invention which produce saintopin derivatives, conventional methods for culturing forms are generally used. As the medium, either a synthetic medium or a natural medium can be used insofar as it appropriately contains carbon sources, nitrogen sources and inorganic substances which can be assimilated by the strains used and the necessary growth and production promoting substances.

As the carbon sources, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses, etc. can be used alone or in combination. In addition, hydrocarbons, alcohols, organic acids, etc. can also be used according to the assimilation ability of microorganisms. As the nitrogen sources, ammonium chloride, ammonium nitrate, ammonium sulfate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, soaking liquid of cereal germ, soybean powder, casamino acid, etc. can be used alone or in combination. If necessary, inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, magnesium phosphate, calcium carbonate, potassium dihydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, copper sulfate, etc. can be added. In addition, trace amounts that promote the growth of the strain used and the production of the Saintopin derivative can also be added to the medium.

As the method for culturing, liquid cultivation, particularly, underwater agitation cultivation is preferably used. Cultivation is carried out at 16 to 37 °C, preferably 25 to 32 °C, and at a pH of 4 to 10, preferably 6 to 8. In general, the desired substance is produced and accumulated in the culture by culturing for one to seven days. To adjust the pH of the medium, aqueous ammonia, ammonium carbonate solution, etc. are used. When the amount of product in the culture reaches the maximum, the cultivation is stopped.

For isolating and purifying the desired substance from the culture, a conventional method for isolating a microbial metabolite from the culture can be used. For example, the culture is separated into culture filtrate and microbial cells by filtration. The microbial cells are extracted with methanol, acetone or the like. Then, the extract is mixed with the culture filtrate, and the resulting mixture is passed through a column of polystyrene adsorbent such as Diaion HP₂₀ (manufactured by Mitsubishi Kasei Corporation) to adsorb the active ingredient, followed by eluting with ethyl acetate, acetone and the like. The eluate is concentrated and the concentrate is subjected to silica gel column chromatography, high performance liquid chromatography and the like to obtain the desired substance. During the cultivation and purification steps, the desired substance can be detected by the UV absorption of the substance.

Examples according to the invention are given below.

Best mode for carrying out the invention example 1

Paecilomyces sp. UOE 1022 (FERM BP-4066) strain was used as the inoculant strain. The strain was inoculated into 300 ml of an inoculant medium having the following composition in a 2-L Erlenmeyer flask and cultured for 48 hours with shaking at 25 ºC.

Composition of the inoculation medium (in 1 l) 5 g peptone (Kyokuto Pharmaceutical Co. Ltd.), 5 g dry yeast "Ebios", 10 g glucose, 200 ml V8 vegetable juice (Campbell Japan), 0.5 g Mg₃(PO₄)₂ 8H₂O (pH before sterilization 6.0)

The resulting inoculum was transferred into 18 L of a fermentation medium with the following composition at the rate of 5% (volume) in a 30-L fermentation vessel, and cultivation was carried out at 25 ºC with stirring and aeration (rotation: 300 rpm, aeration: 18 L/min).

Composition of fermentation medium (in 1 l) 50 g soluble starch, 30 g cereal germ soaking liquid (Japan Maize Products Co., Ltd.), 0.5 g KH₂PO₄, 0.5 g MgPO₄ 7H₂O, 0.5 g Mg₃(PO₄)₂ 8H₂O (pH before sterilization 7.0, adjusted with NaOH)

Cultivation was carried out for 144 hours without controlling the pH of the medium.

After the end of culturing, 7.5 L of the fermentation culture was filtered off, and 10 L of methanol was added to the cell fraction, followed by stirring, thereby extracting UCE 1022. To the methanol extract, 30 L of water was added, and the mixture was passed through a column packed with 0.5 L of a non-ionic porous resin, Diaion HP20 (manufactured by Mitsubishi Kasei Corporation) to absorb the drug. After impurities were eluted with methanol/water (30:70, v/v), the drug was eluted with 1.5 L of methanol/water (50:50, v/v). The drug fraction thus eluted was concentrated, and the concentrate was applied to a column packed with 0.5 L of silica gel (BW 300, manufactured by Fuji Devison Chemical Ltd.), followed by digestion with ethyl acetate/methanol/water (3:2:1, v/v). The drug fraction thus eluted was concentrated, and the concentrate was applied to a column packed with 125 mL of silica gel (Lichroprep-Si 60, Art 9390; manufactured by Merck & Co., Inc.), followed by digestion with ethanol/water (100:3, v/v). The drug fraction thus eluted was concentrated to dryness to give 20 mg of UCE 1022 as a red powder.

Example 2

Paecilomyces sp. SPC-13780 (FERM BP-2256) was used as the seed strain. The strain was inoculated into 300 ml of a seed medium having the following composition in a 2-L Erlenmeyer flask and cultured at 30 ºC for 48 hours with shaking (200 rpm).

Composition of the seed medium (in 11) 5 g peptone, 5 g dry yeast "Ebios", 10 g glucose, 200 ml V8 vegetable juice, 0.5 g Mg₃(PO₄)₂ 8H₂O (pH before sterilization 6.0) The resulting seed culture was transferred into 15 l of a fermentation medium with the following composition at the rate of 5% (volume) in a 30-l fermentation vessel, and cultivation was carried out at 30 ºC with stirring and aeration (rotation: 400 rpm, aeration: 15 l/min).

Composition of the fermentation medium (in 11) 35 g glycerol, 35 g glucose, 15 g dry yeast, 0.5 g KH₂PO₄, 0.5 g MgPO₄ 7H₂O, 0.5 g Mg₃(PO₄)₂ 8H₂O (pH before sterilization 7.0, adjusted with NaOH)

Cultivation was carried out for 80 hours without controlling the pH of the medium.

After filtering the resulting culture, 5 L of acetone was added to the obtained cells, followed by stirring. Removal of the cells and precipitates by filtration yielded 5 L of a filtrate. To the filtrate were added 15 L of ethyl acetate and 0.5 L of ammonia. After stirring, the resulting mixture was passed through a column packed with 2 L of silica gel to adsorb the drug. The drug was eluted with acetone/ethyl acetate/ammonia (1:1:0.05, v/v). The drug fraction was concentrated, and the concentrate was diluted with a 10 mM phosphate buffer (pH 8.0) and passed through a column packed with a polystyrene adsorption resin, Diaion HP20 (manufactured by Mitsubishi Kasei Corporation; 400 mL) to adsorb the drug. After impurities were eluted with a 10 mM phosphate buffer (pH 8.0) and then with 40% methanol containing the buffer at the same concentration, the drug was eluted with 60% methanol.

The drug fraction was concentrated, and the concentrate was diluted with a 10 mM phosphate buffer (13H value 8.0) and subjected to column chromatography using a polystyrene adsorption resin, Diaion HP20SS (manufactured by Mitsubishi Kasei Corporation; 200 mL). The solution eluted with 60% methanol was concentrated, and the concentrate was dissolved in a small amount of a mixture of methanol and acetone. The drug was precipitated from the solution by adding hexane. The precipitate was separated by filtration and dried to give 33 mg of Saintopin E as a bluish-purple powder.

Claims (5)

1. Compound of formula (I) in which R¹ is a hydrogen atom and R² is SO₂OH, or R¹ is an acetyl group and R² is a hydrogen atom. 2. A compound according to claim 1 for use as an antibacterial or antitumor agent. 3. Antibacterial or antitumor composition comprising a compound according to claim 1 as an active ingredient 4. A process for producing a compound according to claim 1, comprising culturing a microorganism of the genus Paecilomyces capable of producing the compound in a culture medium, whereby the product compound can accumulate in the culture, and recovering the accumulated product from the culture. 5. The method according to claim 4, wherein the microorganism used is from the strain SPC-13780 (FERM BP-2256) or from the strain UOE 1022 (FERM BP-4066) or is derived therefrom.